title: Engineering a recyclable elastin-like polypeptide capturing scaffold for non-chromatographic protein purification.
abstract: Previously, we reported a non-chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin-like polypeptide (ELP) to provide fast and cost-effective protein purification. However, the bound dockerin-intein tag cannot be completely dissociated from the ELP-cohesin capturing scaffold due to the high binding affinity, resulting in a single-use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium-coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA-mediated dissociation of the bound dockerin-intein tag from the ELP-cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non-chromatographic based affinity method provides an attractive approach for efficient and cost-effective protein purification.
authors.affiliation: Dept. of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
authors.forename: Wilfred
authors.initials: W
authors.lastname: Chen
mesh_terms: D001709:Biotechnology; D004549:Elastin / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D004549:Elastin
keywords: cohesin-dockerin; elastin-like polypeptide; intein; protein purification; recycle